Unusual suspects: Glial cells in fertility regulation and their suspected role in polycystic ovary syndrome

Abstract Gonadotropin‐releasing‐hormone (GnRH) neurons sitting within the hypothalamus control the production of gametes and sex steroids by the gonads, therefore ensuring survival of species. As orchestrators of reproductive function, GnRH neurons integrate information from external and internal cues. This occurs through an extensively studied neuronal network known as the “GnRH neuronal network.” However, the brain is not simply composed of neurons. Evidence suggests a role for glial cells in controlling GnRH neuron activity, secretion and fertility outcomes, although numerous questions remain. Glial cells have historically been seen as support cells for neurons. This idea has been challenged by the discovery that some neurological diseases originate from glial dysfunction. The prevalence of infertility disorders is increasing worldwide, with one in four couples being affected; therefore, it remains essential to understand the mechanisms by which the brain controls fertility. The “GnRH glial network” could be a major player in infertility disorders and represent a potential therapeutic target. In polycystic ovary syndrome (PCOS), the most common infertility disorder of reproductive aged women worldwide, the brain is considered a prime suspect. Recent studies have demonstrated pathological neuronal wiring of the “GnRH neuronal network” in PCOS‐like animal models. However, the role of the “GnRH glial network” remains to be elucidated. In this review, I aim to propose glial cells as unusual suspects in infertility disorders such as PCOS. In the first part, I state our current knowledge about the role of glia in the regulation of GnRH neurons and fertility. In the second part, based on our recent findings, I discuss how glial cells could be implicated in PCOS pathology.

current knowledge about the role of glial cells in fertility regulation and then discuss new evidence supporting glial cells as potential contributors to PCOS. To finish, I discuss future research avenues.

| INTRODUCTION
The hypothalamic-pituitary-gonadal (HPG) axis regulates the timely production of gametes by male and female gonads essential to the survival of all mammalian species. The hypothalamus, based in the ventral part of the forebrain, is a highly conserved brain structure that integrates information from external and internal cues to orchestrate reproductive outcomes. Specifically, a subset of neurons located in the ventral part of the forebrain, the gonadotropinreleasing-hormone (GnRH) neurons, secrete the neurohormone GnRH. This neurohormone triggers the secretion of gonadotropins by the pituitary that then act on the gonads to produce sex steroids and gametes. Sex steroids feedback to GnRH neurons indirectly via a network of neurons located throughout the hypothalamus referred to as the "GnRH neuronal network." 1,2 However, the brain is not simply composed of neurons.
Glial cells compose at least half of the brain depending on the region analysed. The cortex has a glia-to-neuron ratio (GNR) of 1:1; however, the brainstem, diencephalon and striatum have a GNR closer to 10:1. 3,4 Despite this, their functional role in the central control of fertility remains underappreciated. The classical view of glial cells in the brain is as mere supportive cells for neurons. The latest technological advances in the neuroscience field have led to a wealth of experimental studies demonstrating the functional roles of glial cells in shaping and regulating brain function during development, adulthood and aging. 5,6 In addition, recent studies have revealed that numerous pathologies of the nervous systems target glia. 5,6 Concerning the role of glia in the neuroendocrine regulation of fertility, pioneering studies in the early 1990s highlighted the role of glial cells on reproductive function not only through neuroanatomical studies of the relationship between GnRH neurons and glia, but also through investigation of the role of specific molecules known to be implicated in neuroglial communication. 7 These findings promoted the role of the "GnRH glial network" in sexual maturation throughout postnatal development, as well as in relaying internal and external cues to the GnRH neurons to regulate the timely secretion of GnRH in females during puberty and adulthood. However, to date, numerous questions still remain regarding the specific role of different glial cell types in the control of GnRH neuron activity, GnRH secretion and fertility outcomes. Are all glial cell types associated with GnRH neurons? How are they regulating GnRH neuron activity and secretion? Could they play key roles in infertility disorders such as PCOS?
Outside the scope of fundamental knowledge on how the "GnRH glial network" regulates fertility, investigating the role of glial cells in infertility disorders could lead to the discovery of new targets and treatments in this world of increasing infertility disorders with one in four couples worldwide affected by infertility. 8 Among women with infertility, polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility worldwide, currently affecting 5%-20% of women of reproductive age. 9 As the name indicates, PCOS has long been seen primarily as an ovarian disorder. However, in PCOS patients, elevated luteinising hormone (LH) pulse frequency associated with impaired sex steroid feedback has led to the hypothesis that the communication between the brain and ovaries is impaired. 10,11 Subsequently, evidence from animal-based models of PCOS has highlighted the role of the brain in this disorder and, more particularly, the role of the GnRH neuronal network. 12,13 However, a major neurocentric view of this pathophysiology has contributed to a lack of studies on the role of glial cells in this infertility disorder.
In this review, I aim to propose glial cells as unusual suspects in neuroendocrine infertility disorders such as PCOS. Leading to this idea, the first part of the review briefly develops the mechanism by which the brain regulates fertility and describes the current state of knowledge on the role of different glial cell types in fertility maturation and regulation with a focus on GnRH neuron function. In the second part of this review, I summarise how the communication between the brain and the ovaries is impaired in PCOS and discuss the potential role of some glial cell types in the establishment and maintenance of PCOS pathophysiology.

| DO GLIAL CELLS PLAY A ROLE IN FERTILITY REGULATION?
Fertility is controlled by a small population of brain cells known as GnRH neurons ( Figure 1A). GnRH neurons secrete pulses of GnRH peptide into the pituitary portal system to drive the pulsatile release of LH and follicle-stimulating hormone (FSH) from the pituitary gland.
LH and FSH then act on the gonads to stimulate the production of gametes and sex steroid hormones in a sexually dimorphic manner. In females, once per reproductive cycle, GnRH secretion is increased in a surge pattern that triggers a preovulatory LH surge leading to ovulation. Circulating concentrations of sex steroid hormones are then conveyed to GnRH neurons; however, GnRH neurons do not express the sex steroids receptors that are essential for the feedback regulation of the HPG axis. 1,2 Positive and negative sex steroid hormone feedback has been shown to occur in part through a synaptically-connected neuronal network commonly referred to as the 'GnRH neuronal network'. 1,2 In addition, evidence supports an important role for glial cells in the relay of sex steroid feedback onto GnRH neurons. Sex hormones influence glial cell morphology, number and function throughout life in different brain regions. 14 However, whether or not glial cells express sex steroid receptors depends on the glial cell type, the brain region and the physiological or pathological state of the animal studied. 14,15 In addition, glial cells have been shown to change their morphology and their secretome depending on external (light/season) and other internal (energy balance) cues, as well as to interact/ communicate with GnRH neurons, therefore positioning them as important modulators of the central regulation of the HPG axis ( Figure 1A).

| Glial cells within the brain
Glial cells consist of radial glia, microglia, astrocytes, tanycytes and oligodendrocytes. 5 The radial glia are the progenitors of the central nervous cells generating astrocytes and neurons within specialised neurogenic zones. 5 Astrocytes, the star-shaped cells of the brain, originate from the same neuroepithelial progenitors as neurons and share similar functions, such as expressing G-protein coupled receptors (neuropeptides, neurotransmitter and hormones receptors) 15,16 and signalling molecules, capable of calcium signalling and releasing gliotransmitters. 5,15,17 Astrocytes are mostly known to regulate neuronal activity through the concept of a tripartite synapse, such that they can modulate glutamate release and uptake within the synaptic cleft, therefore modulating synaptic transmission. 5,18 In addition, they have been shown to play a major role in remodelling neuronal circuitry during development and adulthood, being implicated in many relevant aspects of neuronal function, such as neuronal trophic support, control of ion homeostasis, neuronal survival and differentiation, neuronal guidance, neurite outgrowth, and synaptic efficacy. 5,15,17 Tanycytes are a specialised type of ependymocytes lining the ventral part of the third ventricle. Tanycytes are currently classified into four subtypes: α1, α2, β1 and β2, depending on their dorsoventral locations within the third ventricular wall. However, with the advances in technology, new evidence from structural, functional and transcriptomic studies calls for a novel classification closer to their specific functions. 19 Microglia are the resident immune cells of the brain. They originate outside the brain and populate the brain during early embryonic development. 20 Microglia were first investigated with respect to their role as resident immune cells of the adult brain that phagocytose cell debris and recruit macrophages from the periphery during episodes of infections or brain insults such as stroke and ischemia, or in neurodegenerative diseases. 20 However, it is now well established that microglia have a myriad of other functions that shape and maintain homeostasis in the brain.
Microglia are now known for their active role in regulating neuronal wiring throughout development: from the induction of neuronal spine formation to their role in pruning and refining synapses during early development, contributing to the maturation of neuronal circuitries. 20 In the hypothalamus, VanRyzin et al. 21 showed that microglia are regulated by sex steroids and play a role in masculinising the rodent brain.
Oligodendrocytes are the "Schwann cells" of the central nervous system producing myelin sheath for neurons of the central nervous system. 5 Myelin forms an insulating layer around nerve terminals and is implicated in homeostasis and speeding-up the electrical impulses for an efficient communication between central nervous system neurons and the peripheral nervous system. , blocking inputs (black); however, preceding the preovulatory GnRH surge, glial processes allow the GnRH neuronal networks inputs to connect to GnRH neurons, therefore triggering a GnRH surge. (C) Role of glial cells in the vicinity of GnRH nerve terminals: during the basal pulsatile GnRH secretion phase, glial cells (blue), mostly tanycyte end-feets, enwrap GnRH nerve terminals (red) to block their access to portal blood capillaries (brown); however, at the time of the preovulatory surge, tanycyte endfeets retract, allowing GnRH nerve terminals to access the portal blood capillaries, leading to high secretion of GnRH (i.e., the preovulatory surge). GnRH-N, gonadotropin-releasing hormone neurons 2.2 | From neuroanatomical evidence to a suggested functional role in fertility regulation Microglia and oligodendrocytes are the forgotten glial cells when it comes to the GnRH neurons and fertility regulation. For microglia, to date, only two publications have shown microglia association with GnRH soma. 22,23 The first paper is a qualitative study observing microglia in association with GnRH neuron soma of adult female rats. 23 The second paper is our recent study highlighting that, in addition to the presence of microglia in the vicinity of GnRH neurons, their filopodia ensheath GnRH neurons during postnatal development. 22 Concerning oligodendrocytes, the recently published study by Pellegrino et al. 24 showed, for the first time By contrast, astrocytes and tanycytes are well documented glial cell types in terms of proximity to GnRH neuron soma and terminals.
They are also the most extensively studied for their role in regulating GnRH neuron activity, secretion and reproductive outcomes. Evidence supporting the physical and chemical interactions between GnRH neurons and these two glial cell types is discussed below.

| Glial plasticity in the vicinity of GnRH neurons
Coverage of GnRH neuron cell bodies by glial cells, identified by glial fibrillary acidic protein (GFAP), is dependent on developmental age 25,26 and hormonal status in different species. 27,28 In adult female monkeys, a comparison of the GFAP-immunoreactive positive (GFAP-IR + ) coverage at different stages of the menstrual cycle or by artificial treatment with sex steroids (ovariectomy with or without estradiol replacement), suggests that GFAP-IR + cells are influenced by sex steroid levels and can change their morphology accordingly. 28 Interestingly, in their study, Witkin et al. 28  by Vincent Prevot showed that tanycytes are essential components of this structural plasticity in rats, 33 as well as in humans. 34,35 During the luteal phase, tanycytic end-feet have been shown to physically restrict access of the GnRH nerve terminals to the portal blood vessels ( Figure 1C). Then, at the time of the preovulatory surge, tanycytes physically allow GnRH nerve terminals to contact the portal blood vessels by retraction of their end-feet ( Figure 1C). This structural plasticity has also been shown to be dependent upon estrogen signalling. 36  but also through semaphorin-7A and its receptor plexinC1 on GnRH neurons and β1-integrin on tanycyte end-feet. 43 In conjunction, tanycyte-endothelial communication occurs through nitric oxide and growth factors dependent upon estrogen signalling. 36,47 Second, hypothalamic astrocytes and tanycytes have been shown to produce several growth factors such as transforming growth factor (TGF)β, insulin-like growth factor-1 and epidermal growth factor-like peptides (TGFα and neuregulin1) during puberty and adulthood, which then regulate GnRH neuron activity and secretion (Figure 2). 7 The first evidence for this arose from studies in which applications of TGFα to ex vivo median eminence explants was found to increase GnRH release into the culture medium. 48 Thereafter, Ojeda et al. 49

| DO GLIAL CELLS PLAY A ROLE IN THE NEUROENDOCRINE PATHOLOGY OF PCOS?
3.1 | PCOS: A miscommunication between the brain and the ovaries PCOS is the most common anovulatory infertility disorder, affecting between 5% and 20% of women of reproductive age worldwide. 9 PCOS is characterised by the presence of at least two of three diagnostic criteria: elevated androgen hormones, menstrual dysfunction and multiple cyst-like follicles in the ovary. 56 In PCOS patients, LH pulse frequency, which mirrors GnRH neuron activity, is significantly increased. 57 Clinical data have demonstrated impaired estrogen and progesterone feedback in PCOS patients. 10,11 This clinical presentation suggests the brain is a major culprit of PCOS pathology (Figure 4).
Animal-based models of PCOS have helped us to understand the underlying mechanism of this impaired sex steroid feedback. 12

| Unusual suspect: Potential role of glial cells in PCOS
As previously detailed, glial cells are now recognised as major players in brain development and brain function, as well as for playing a major role in disease onset and progression. 5,6 Here, my working hypothesis is that, in PCOS, not only the "GnRH neuronal network", but also the Transient depletion of microglia around P11 has been shown to impair sex steroid-mediated feedback, pubertal onset and oestrus cyclicity, 69 suggesting that microglia are required for establishing normal endocrine function in adulthood. In a recent study, using immunohistochemistry, we compared microglia number and phenotype within two regions of the hypothalamus in the PNA mouse model of PCOS and health controls throughout development and in adulthood. 22 We found a decrease in the total number of microglia in PNA mice at P25, before disease onset ( Figure 5A) and in alignment with the observation of increased GABAergic inputs. This decrease was restricted to the thick microglia phenotype, which is known to be a phagocytic phenotype. 20 Phagocytic function of microglia was then observed during the well-characterised intense synapse pruning period that occurs between P7 and P15 in the mouse. Our findings suggested an impaired phagocytic activity specifically targeted to GABA inputs in the rostral POA with microglia pruning fewer GABAergic terminals at P15 in the PNA mouse model of PCOS ( Figure 5A). 22 These results are in accordance with a recent study using in vivo imaging to demonstrate that microglia interact with cortical GABAergic synapses, around P12-P17, to phagocytose and thus refine GABAergic inputs. 70 Therefore, these findings support an important role for microglia in the onset of prenatal androgen mediated PCOS-like features.

| Do microglia play a role in adult PCOS pathology?
Microglia are the resident immune cells of the brain and therefore their major role in adulthood is to survey the brain parenchyma and trigger inflammatory responses after a pathogen's invasion or any insults within the brain. 20  In PCOS, an increase pulsatile secretion of luteinising hormone (LH) and lower secretion of follicle-stimulating hormone (FSH) has been observed, mirroring a hyperactivity of gonadotropin releasing hormone neurons (GnRH-N) (red), leading to the three cardinal features of PCOS: high circulating level of androgens, polycystic ovaries and oligo/anovulation. Impaired sex hormone feedback has also been observed in patients with PCOS. Animal-based models have allowed to investigate the role of the brain in this disruption of the HPG axis. It has been observed that GABA neurons coming from the arcuate nucleus (ARN GABA-N, green) are less sensitive to at least one sex hormone and send more projections to the GnRH neurons, leading to the hypersecretion of GnRH/LH. We now wonder whether glial cells (blue) could play a major role in the PCOS pathophysiology. ARN GABA-N, GABA-N located in the arcuate nucleus regulation of fertility in females. 71 Clinical and pre-clinical evidence suggests that PCOS may be associated with an inflammatory state. in the ERBB4 loci. 82 As highlighted in the first part of this review, a deletion of ErbB4 in GFAP-expressing cells drive delayed puberty onset, impaired sex steroid feedback and fertility outcomes. 50,83 Therefore, a miscommunication between GnRH and astrocytes through impaired ErbB signalling remains to be investigated in PCOS animal models. Third, microglia and astrocytes have been shown to bi-directionally communicate together, which determines the functional state of each cell type and can lead to central nervous system disease when disrupted. 92 Interestingly, by immunohistochemistry, we observed a close association between microglia and astrocytes in the vicinity of GnRH soma (Desroziers E, unpublished data) (Figure 3). This observation suggests a potential astrocyte-microglia crosstalk dysfunction that could play a role in PCOS pathophysiology.
Finally, as discussed in the first part of this review, the first study showing oligodendrocytes in close association with GnRH neurons has been published and has reported observing oligodendrocytes within the vicinity of GnRH soma in the brain of juvenile rats. 24 Interestingly, it has been observed that women affected by multiple sclerosis (MS), a demyelinating disease, are also affected with reproductive dysfunction, such as by an increase in serum levels of LH and FSH, a decreased serum estrogen concentration, menstrual irregularities and infertility. 93 This indicates that degeneration of the myelin sheath in the brain could impact neurons regulating fertility. Noteworthy, MS is also an autoimmune disorder where the immune system attacks the neurons and is responsible for the demyelination of the nerve terminals and, as such, it cannot be ruled out that the resident immune cells of the brain, microglia, could also be the culprit in the infertility observed in women with MS. Therefore, it would be of interest to deepen the investigation of the role of oligodendrocytes in GnRH function, fertility regulation and PCOS pathophysiology. has shown a role for microglia in the establishment of PCOS pathology, although other glial cell types could also be culprits. By investigating the role of glial cells in PCOS pathophysiology, we will increase our understanding of PCOS disease mechanisms and potentially uncover future avenues with respect to prevention and treatment.

ACKNOWLEDGMENTS
My biggest and infinite thanks go to my mentor P. Rebecca Campbell not only for the feedback she provided on this review, but also for her unconditional support over the last 6 years, which has been instrumental in developing my independent career. Thanks also to Aisha Sati, PhD student, not only for providing feedback on this manuscript, but also most importantly for her astonishing work on the role of microglia in the PNA

CONFLICTS OF INTEREST
The author declares that she has no conflicts of interests.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this review because no new data were created or analysed.